The invention pertains to the field of molecular biology and techniques and apparatus for the manufacture of DNA molecules of defined or desired sequences. The er vivo manufacture of DNA molecules makes possible the use of those DNA molecules in vivo to synthesize any desired peptides, proteins or assemblies of proteins or combinations of nucleic acids, as may be desired, and to perform a large variety of genetic experiments in living organisms.
In modern biotechnology it is common to create DNA sequences chemically, that is to say apart from any living organism. The DNA sequences are assembled and replicated in vitro using cell free techniques and ultimately are recombined or reassembled into DNA sequences which can be inserted into organisms for biological purposes. It has become commonplace to synthesize short DNA sequences, referred to as oligonucleotides, directly from individual nucleosides and to construct larger DNA sequences from smaller oligonucleotides.
It has also been proposed that one may create larger DNA molecules by the making many smaller, but properly designed, DNA molecules in parallel, and then permitting those smaller molecules, or fragments, to self-assemble and thereby make a longer oligonucleotide. This can be done most conveniently by using a maskless array synthesizer (MAS) instrument, of the type disclosed in published PCT application WO 99/42813 to create a number of single stranded DNA sequences in a massively parallel synthesis operation. The single stranded DNA sequences thus created can then be cleaved from the substrate upon which they are constructed and permitted to anneal and form much larger DNA segments. This process and the general principles behind its operation are described in published PCT application PCT/US02/15951, the disclosure of which is incorporated herein by reference. A unique attribute of the MAS instrument of particular interest for this process is that fact that the instrument can create microarrays in which the oligonucleotides attach in the array are quite long, as long as 60 to 100 nucleotides. It is believed that only this style of microarray instrument permits the synthesis of oligonucleotides of this length in a microarray.
While these methods permit the synthesis of long DNA segments, using the massively parallel synthesis capabilities of the MAS style of instrument, there is one drawback to the methodology described in the aforementioned published PCT applications. The amount of DNA assembled by this synthesis, without other procedures, is relatively small. While the amount of DNA is so small as to be difficult to measure physically, it is believed that approximately 20 picomoles of DNA are synthesized on a typical DNA microarray made by an MAS instrument. The fact that the amount of DNA is this small makes the physical handling and amplification of the DNA a relatively sophisticated procedure. The DNA is so small an amount that if the DNA is handled at a concentration consistent with most DNA reaction conditions, the volume becomes much smaller than the volumes suitable for handling in most fluid handling operations. Conversely, if the solution is diluted to a reasonable volume, then the DNA molecules are so dilute in the solution that the normal enzymes and other agents used for altering and manipulating DNA are difficult to use because of the dilution of the DNA. Accordingly, the process for the massive parallel synthesis of DNA fragments would be improved if methodologies existed to dramatically increase the amount of DNA which is the product of such a process.